Pecan (Carya illinoinensis) is an important tree nut throughout the world. The high concentration of flavonoid in its kernels makes it an excellent food with health benefits. However, the molecular basis of flavonoid biosynthesis in pecan remains unclear, which hinders quality breeding in this plant. Therefore, in order to find the crucial genes involved in flavonoid biosynthesis, the changes in flavonoid profiles and the transcriptomes of pecan kernels at four developmental stages (late water, gel, dough, and mature stages) were analyzed. As a result, the highest levels of total phenolic, condensed tannin, and flavan-3-ols were observed at the "late water stage". Catechin was the most abundant flavan-3-ol at different development stages. In total, 64 773 unigenes were obtained, and 46 924 (72.44%) unigenes were annotated. After differentially expressed gene (DEG) analysis, 12 750 unique DEGs were identified. Flavonoid-related DEGs of 36 structural genes and eight MYBs were obtained. The structural gene set contained three PALs, three CHSs, two CHIs, one F3H, two F3′Hs, two F3′5′Hs, one DFR, one ANS, two LARs, and two ANRs. The expression patterns of most of the structural genes were consistent with the changes in flavonoid profiles during kernel development. We believe that this RNA-Seq data set will provide valuable resources for unraveling the molecular mechanism of flavonoid metabolism in pecan and will significantly promote genetic studies and quality breeding in this plant.
Bioengineering of
ribosomally synthesized and post-translationally
modified peptides (RiPPs) is an emerging approach to explore the diversity
of pseudo-natural product structures for drug discovery purposes.
However, despite the initial advances in this area, bioactivity reprogramming
of multienzyme RiPP biosynthetic pathways remains a major challenge.
Here, we report a platform for de novo discovery of functional thiopeptides
based on reengineered biosynthesis of lactazole A, a RiPP natural
product assembled by five biosynthetic enzymes. The platform combines
in vitro biosynthesis of lactazole-like thiopeptides and mRNA display
to prepare and screen large (≥1012) combinatorial
libraries of pseudo-natural products. We demonstrate the utility of
the developed protocols in an affinity selection against Traf2- and
NCK-interacting kinase (TNIK), a protein involved in several cancers,
which yielded a plethora of candidate thiopeptides. Of the 11 synthesized
compounds, 9 had high affinities for the target kinase (best K
D = 1.2 nM) and 10 inhibited its enzymatic activity
(best K
i = 3 nM). X-ray structural analysis
of the TNIK/thiopeptide interaction revealed the unique mode of substrate-competitive
inhibition exhibited by two of the discovered compounds. The thiopeptides
internalized to the cytosol of HEK293H cells as efficiently as the
known cell-penetrating peptide Tat (4–6 μM). Accordingly,
the most potent compound, TP15, inhibited TNIK in HCT116 cells. Altogether,
our platform enables the exploration of pseudo-natural thiopeptides
with favorable pharmacological properties in drug discovery applications.
We
report a method for the high-throughput reactivity profiling
of genetically encoded libraries as a tool to study substrate fitness
landscapes for RiPP (ribosomally synthesized and post-translationally
modified peptide) biosynthetic enzymes. This method allowed us to
rapidly analyze the substrate preferences of the lactazole biosynthetic
pathway using a saturation mutagenesis mRNA display library of lactazole
precursor peptides. We demonstrate that the assay produces accurate
and reproducible in vitro data, enabling the quantification of reaction
yields with temporal resolution. Our results recapitulate the previously
established knowledge on lactazole biosynthesis and expand it by identifying
the extent of substrate promiscuity exhibited by the enzymes. This
work lays a foundation for the construction and screening of mRNA
display-based combinatorial thiopeptide libraries for the discovery
of lactazole-inspired thiopeptides with de novo designed biological
activities.
Single molecule dynamics studies have begun to use quantum probes. Single particle analysis using cryo-transmission electron microscopy has dramatically improved the resolution when studying protein structures and is shifting towards molecular motion observations. X-ray free-electron lasers are also being explored as routes for determining single molecule structures of biological entities. Here, we propose a new X-ray single molecule technology that allows observation of molecular internal motion over long time scales, ranging from milliseconds up to 103 seconds. Our method uses both low-dose monochromatic X-rays and nanocrystal labelling technology. During monochromatic X-ray diffraction experiments, the intensity of X-ray diffraction from moving single nanocrystals appears to blink because of Brownian motion in aqueous solutions. X-ray diffraction spots from moving nanocrystals were observed to cycle in and out of the Bragg condition. Consequently, the internal motions of a protein molecule labelled with nanocrystals could be extracted from the time trajectory using this diffracted X-ray blinking (DXB) approach. Finally, we succeeded in distinguishing the degree of fluctuation motions of an individual acetylcholine-binding protein (AChBP) interacting with acetylcholine (ACh) using a laboratory X-ray source.
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